1. Field of the Invention (Technical Field)
The present invention relates to an apparatus for processing multiple plug samples in an automated fashion for flow cytometry, using a plurality of sample loops, a reciprocating valve, and at least one syringe or pump. The present invention is also a method for rapidly processing multiple samples in flow cytometry.
2. Background Art
Flow cytometry is a tool frequently utilized for cellular analysis. In flow cytometry, a sample containing cells (or equivalent particles) is passed through a line with a sheath fluid, and focused through an orifice, with the objective of cells entering the detector one at a time. These individual cells are irradiated with a light beam, and the intensity of scattered light or fluorescent light from the cells is measured. The ability of flow cytometry to make quantitative measurements on microspheres or cells with continuous kinetic resolution offers an attractive platform for more detailed characterization of compounds of interest.
Traditionally, samples are injected into and suspended within a sheath flow. This type of delivery is disclosed in the following patents: U.S. Pat. No. 5,824,269, to Kosaka et al., entitled Flow Cytometer; European Patent No. 46,345, to Connell, entitled Controlled Hydrodynamic Flow in Flow Cytometry Systems; U.S. Pat. No. 5,374,398, to Isami et al., entitled Apparatus for Analyzing Particles; U.S. Pat. No. 5,369,137, to Hansen, entitled Simultaneous Multiple Assays; and U.S. Pat. No. 5,286,452, to Hansen entitled Simultaneous Multiple Assays. The articles Hodder, P. et al., Microfabricated Flow Chamber for Fluorescence-based Chemistries and Stopped-flow Injection Cytometry, Analyst, 122:883-887 (1997); and Nolan, J. et al., A Rapid Mix Flow Cytometer with Subsecond Kinetic Resolution, Cytometry, 21(3):223-229 (1995), disclose the use of syringe pumps for a stopped-flow injection analysis. The sample stream is bounded by the reagent stream. A stopping of the sheath flow causes the core to expand the sheath, thus mixing the sample with the reagent. These inventions do not "compartmentalize" the sample, or prevent mixing. Lack of mixing is especially desirable when screening a multitude of samples, in order to prevent the possibility of cross-contamination.
One rapidly expanding field for which such cellular information is useful is that of drug discovery. Large combinatorial libraries that potentially alter or mimic receptor-ligand interactions are screened. To efficiently perform such tasks, rapidity and automation of sample handling are required.
Various devices have been developed to address sample handling. These include use of a packet type of sample delivery. U.S. Pat. No. 5,268,147, to Zabetakis et al., entitled Reversible Direction Capsule Chemistry Sample Liquid Analysis System and Method, discloses supply tubes for samples, reagents, buffers, and isolation liquid. The device functions with a positive displacement pump. Air is introduced at intervals to separate the samples. This does not result in a continuous, uninterrupted flow. Samples are not loaded while others are transferred, thus increasing processing time. U.S. Pat. No. 4,853,336, to Saros et al., entitled Single Channel Continuous Flow System, discloses sample segments separated by immiscible segments in a single conduit, which are then removed to combine and mix. While this is a continuous flow system, the goal of this invention is mixture of the sample prior to sampling. A major problem with introducing air bubbles or mixing, however, is the potential of the bubbles lodging in troublesome areas such as the analysis region of the flow cell. Also, air bubbles can be broken up and dispersed at connector junctions, causing inaccuracies in the sample draw.
Another method developed to increase sample delivery rate is the use of loops. Inventions containing loops include the following: U.S. Pat. No. 5,788,927, to Farrell, et al., entitled Unified Fluid Circuit Assembly for a Clinical Hematology Instrument, utilizes loops that align with the reagent passageways, but not samples. In Farrell, the shear valve is rinsed after each sample, so it does not disclose continuous sampling. U.S. Pat. No. 4,957,009, to Nohl et al., entitled Pushloop Liquid Sampling Method, discloses a six-port valve which pulls a sample past a loop, then pushes it back into the chamber. This device, as well, includes a flushing or rinsing step, and therefore is not a continuous delivery device. U.S. Pat. No. 4,224,033, to Hansen et al., entitled Programmable, Continuous Flow Analyzer, discloses utilizing valves to switch loops in order to add samples and reagents to carriers. This method involves inserting the samples within the carriers and subsequently mixing the sample with the carrier.
Yet another approach is the use of valves to increase sample processing speed. Such inventions include the following: U.S. Pat. No. 3,921,439, to Burns, entitled Method and Apparatus for Selectively Removing Immiscible Fluid Segments from a Fluid Sample Stream, discloses a series of valves within conduits. The function of these valves is to remove immiscible fluid segments prior to sampling. Therefore, they do not contribute to the sample, but rather function to take away from the sample. U.S. Pat. No. 5,776,781, to Vardanega et al., entitled Sterile Flow Cytometer and Sorter with Mechanical Isolation between Flow Chamber and Sterile Enclosure and Methods for Using Same, and U.S. Pat. No. 5,641,457, to Vardanega et al., entitled Sterile Flow Cytometer and Sorter with Mechanical Isolation between Flow Chamber and Sterile Enclosure, disclose injecting cells into a sheath fluid by the force of air pressure. The valves are used to regulate flow, and convert operating mode from back-flush to sort, etc. U.S. Pat. No. 5,395,588, to North, Jr. et al., entitled Control of Flow Cytometer Having Vacuum Fluidics, discloses a device that utilizes a pump to pull samples. It has four normally closed solenoid actuated valves, two of which open to the sample, and two of which open to drain the device. They are not reciprocating. U.S. Pat. No. 5,488,469, to Yamamoto, et al., entitled Cell Analyzing Apparatus, discloses a device that uses cell fluorescence analysis. It has a sample pump to draw the sample out of the tube, and a three-way valve which switches to allow the sheath fluid to be pumped past the sample, drawing it into the feed tube. These inventions utilize either air pressure or a pump to pull the sample, which may cause problems with sample handling such as creation of air bubbles and the need to flush the system between the samples.
Still a further approach to automate sample handling is described in U.S. Pat. No. 4,177,677, to Ruzicka et al., entitled Sample Supply to Automatic Analyzers, disclosing a valve which couples a sample conduit to a sample and, alternatively, to a carrier stream leading to an analytical device. The sample is pumped up to the sample conduit, then back through the carrier conduit to the analyzer. This device allows for samples of 30 .mu.l or more to be processed, but is limited to processing one sample draw at a time.
Other inventions which disclose injecting the sample or another fluid, but lack the other aspects involved in the present invention (i.e. plugs and reciprocating valves), include U.S. Pat. No. 5,464,752, to Kortright, et al., entitled Automated Analyzer for Screening Cells or Formed Bodies for Enumeration of Populations Expressing Selected Characteristics; U.S. Pat. No. 5,221,521, to Hashizumi et al., entitled Sample Liquid Dilution System for Analytical Measurements; U.S. Pat. No. 5,080,866, to Petty et al., entitled Analytic Apparatus and Method; and U.S. Pat. No. 4,399,225, to Hansen et al., entitled Stop-Flow Analysis.
None of the previous inventions are capable, due to design constraints, of processing multiple samples in a fashion rapid enough to effectively screen large libraries. To address this need, the present invention is a novel approach to automated sample handling, disclosing a continuously flowing steam of fluid into which individual samples are sequentially inserted as a bolus or "plug" of precisely defined volume. The stream delivers the sample plugs, separated by empty volumes of fluid (the buffer of which the stream is composed), to the point of analysis in the laser beam. Because an automated syringe was used for sample uptake, pressurization of the sample-containing vessel was unnecessary. This system leads to faster sampling rates and smaller required sample volumes.